Methods for treating intrapulmonary infections

ABSTRACT

This disclosure relates to the treatment of intrapulmonary bacterial infections, including treatment of nosocomial pneumonia lung infections with pharmaceutical compositions containing the cephalosporin ceftolozane.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/607,138, filed Sep. 7, 2012, which claims priority to U.S.Provisional Application No. 61/532,914, filed Sep. 9, 2011, titled“Methods for Treating Intrapulmonary Infections,” and U.S. ProvisionalApplication No. 61/657,386, filed Jun. 8, 2012, titled “Methods forTreating Intrapulmonary Infections.” The contents of any patents, patentapplications, and references cited throughout this specification arehereby incorporated by reference in their entireties.

TECHNICAL FIELD

This disclosure relates to the treatment of intrapulmonary bacterialinfections, including the treatment of nosocomial pneumonia infections,with a cephalosporin.

BACKGROUND

The cephalosporin(6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureido]-1-methyl-1H-pyrazol-2-ium-2-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(Z)-1-carboxy-1-methylethoxyimino]acetamido]-3-cephem-4-carboxylicacid (also referred to as “CXA-101” and previously designated FR264205)is an antibacterial agent. CXA-101 can be provided as the compound shownin FIG. 1. The antibacterial activity of CXA-101 is believed to resultfrom its interaction with penicillin binding proteins (PBPs) to inhibitthe biosynthesis of the bacterial cell wall which acts to stop bacterialreplication. CXA-101 can be combined (e.g., mixed) with a β-lactamaseinhibitor (“BLI”), such as tazobactam. Tazobactam is a BLI against ClassA and some Class C β-lactamases, with well established in vitro and invivo efficacy in combination with active β-lactam antibiotics. Thecombination of CXA-101 and tazobactam in a 2:1 weight ratio is anantibiotic pharmaceutical composition (“CXA-201”) for parenteraladministration. CXA-201 displays potent antibacterial activity in vitroagainst common Gram-negative and selected Gram-positive organisms.CXA-201 is a broad-spectrum antibacterial with in vitro activity againstEnterobacteriaceae including strains expressing extended spectrumβ-lactamases-resistant (MIC₉₀=1 mg/mL), as well as Pseudomonasaeruginosa (P. aeruginosa) including multi-drug resistant strains(MIC₉₀=2 μg/mL). CXA-201 is a combination antibacterial with activityagainst many Gram-negative pathogens known to cause intrapulmonaryinfections, including nosocomial pneumonia caused by P. aeruginosa.

Intrapulmonary infections, such as nosocomial pneumonia, remain a majorcause of morbidity and mortality, especially infections caused by drugresistant pathogens such as P. aeruginosa. One challenge in treatingintrapulmonary infections with systemic administration of an antibioticis determining the antibiotic dose that will provide a therapeuticallysafe and effective concentration of the antibiotic at the site of aninfection on the mucosal side of the bronchi in the lung (i.e., in thebronchial secretions). Many antibiotics diffuse poorly from thebloodstream across the bronchi [e.g., Pennington, J. E., “Penetration ofantibiotics into respiratory secretions,” Rev Infect Dis 3(1):67-73(1981)], which can result in the administration of higher doses ofantibiotic than would be prescribed for a truly systemic infection.Furthermore, the purulent sputum that characterizes infected patientstends to compromise the potency of many antibiotics (See e.g., Levy, J.,et al., “Bioactivity of gentamicin in purulent sputum from patients withcystic fibrosis or bronchiectasis: comparison with activity in serum,” JInfect Dis 148(6):1069-76 (1983)). In some cases, the result is theprescription of large amounts of a systemically administered antibioticto treat an intrapulmonary infection.

The efficacy of an antibiotic depends in part on the concentration ofthe drug at the site of action. Efficacy of antimicrobial therapyrequires adequate antibiotic concentrations at the site of bacterialinfection, and some authorities believe that epithelial lining fluid(ELF) concentrations are a reasonable surrogate for predicting effectiveconcentrations for treating intrapulmonary infections such as pneumonia.For many antibiotics, clinical data correlating ELF concentrations toclinical outcome is unavailable and the clinical significance ofdifferences in pulmonary penetration of antibiotics is unknown or poorlycharacterized. Few studies have quantified the penetration of β-lactamagents into the lung, as measured by the ratio of area under theconcentration-time curve (AUC) in ELF to AUC in plasma(AUC(ELF)/AUC(plasma) ratio). For some published studies, theconcentration of antibiotics measured in the ELF of the lung has variedwidely. For example, the reported penetration ratio of telavancin inhealthy human volunteers ranges widely between 0.43 and 1.24 (Lodise,Gottfreid, Drusano, 2008 Antimicrobial Agents and Chemotherapy). Thus,predicting the penetration of a drug into the ELF a priori, based on thestructure, molecular weight, size and solubility is difficult due to thelimited data available on the effect of physicochemical properties onthe lung penetration of drugs.

Accordingly, the efficacy of a particular drug in treatingintrapulmonary infections, in particular nosocomial pneumonia, cannot bepredicted solely on the basis of data, such as in vitro data relating tothe activity of that drug against a particular bacterium, which does notgive any indication as whether the drug will accumulate at atherapeutically safe and effective concentration at the site of aninfection on the mucosal side of the bronchi in the lung (i.e., in thebronchial secretions). For instance, tigicycline, a glycylcyclineantimicrobial, has in vitro activity against many species ofGram-positive and Gram-negative bacteria, including P. aeruginosa, andit has been approved by the FDA for the treatment of complicated skinand skin structure infections, complicated intra-abdominal infections,and community acquired pneumonia. However, tigicycline is not approvedfor the treatment of nosocomial pneumonia, in view of an increasedmortality risk associated with the use of tigicycline compared to otherdrugs in patients treated for nosocomial pneumonia.

SUMMARY

The present invention provides methods for treating intrapulmonaryinfections, including nosocomial pneumonia, with systemic administrationof a pharmaceutical composition comprising ceftolozane. The invention isbased in part on results from a human clinical study designed to assessthe ELF penetration of CXA-201 in comparison to piperacillin/tazobactam,indicated for the treatment of nosocomial pneumonia. The study describedherein quantified the penetration of CXA-201 into the lung, as measuredby the ratio of area under the concentration-time curve (AUC) inepithelial lining fluid (ELF) to AUC in plasma (AUC(ELF)/AUC(plasma)ratio). The results of the study indicate that CXA-201 penetrated intothe ELF of human patients, with a ceftolozane ELF/plasma AUC ratio of0.48. The measured ELF concentrations of ceftolozane exceeded 8 μg/mLfor 60% of the 8-hour dosing interval, a concentration that is predictedto inhibit 99% of Pseudomonas aeruginosa based on current surveillancedata.

The study showed that CXA-201 penetrated well into the ELF of healthyvolunteers compared to piperacillin/tazobactam, an agent widely used fortreatment of lower respiratory infections. The intrapulmonarypharmacokinetics measured in the study supports the use of CXA-201 as aparenteral (e.g., intravenous) antibiotic for treatment ofintrapulmonary infections, such as nosocomial pneumonia or other lowerrespiratory tract infections.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is the chemical structure of a salt of ceftolozane hydrogensulfate salt.

FIG. 2A is a graph showing the ELF Concentration vs. Time Profile forceftolozane hydrogen sulfate salt (Median and Range) for CXA-201.

FIG. 2B is a graph showing the ELF Concentration vs. Time Profile forTazobactam (Median and Range) for CXA-201.

FIG. 3A is a graph showing the (Comparative) ELF Concentration vs. TimeProfile for Piperacillin (Median and Range) for apiperacillin/tazobactam comparator (ZOSYN®).

FIG. 3B is a graph showing the (Comparative) ELF Concentration vs. TimeProfile for Tazobactam (Median and Range) for a piperacillin/tazobactamcomparator (ZOSYN®).

FIGS. 4A and 4B are synthetic schemes for preparing ceftolozane hydrogensulfate salt.

DETAILED DESCRIPTION

The present disclosure relates to the treatment of intrapulmonaryinfections, including nosocomial pneumonia, with systemic administrationof a pharmaceutical composition comprising ceftolozane, including theparenteral administration of a therapeutically effective amount of apharmaceutical composition comprising ceftolozane and tazobactam. Asused herein, the term “ceftolozane” means CXA-101 in a free-base or saltform, preferably a hydrogen sulfate form (illustrated in FIG. 1). In oneembodiment, ceftolozane is CXA-101 in its free-base form. In anotherembodiment, ceftolozane is CXA-101 in its salt form, preferably ahydrogen sulfate form.

In a preferred embodiment, ceftolozane (in free base or salt form,preferably hydrogen sulfate form) and tazobactam are in a 2:1(ceftolozane:tazobactam) weight ratio. In a particular embodiment,provided herein are methods of treating intrapulmonary infections,including nosocomial pneumonia, with systemic administration of apharmaceutical composition comprising ceftolozane hydrogen sulfate andtazobactam in a 2:1 weight ratio. The combination of ceftolozanehydrogen sulfate and tazobactam in a 2:1 weight ratio is referred toherein and in the examples as “CXA-201.”

In one aspect, the invention provides a method of treating anintrapulmonary infection comprising administering a therapeuticallyeffective amount of a pharmaceutical composition comprising ceftolozane.The method may comprise administering a pharmaceutical compositioncomprising ceftolozane in combination with tazobactam.

In another aspect, the invention provides a method of treating anintrapulmonary infection comprising the step of intravenouslyadministering about every 8 hours to a subject in need thereof apharmaceutical composition comprising 3.0 g of ceftolozane. The methodmay comprise administering a pharmaceutical composition comprisingceftolozane in combination with tazobactam. In one embodiment, themethod comprises administering CXA-201 and the infection comprisesGram-negative bacteria. In another aspect, the invention provides amethod of treating an intrapulmonary infection comprising the step ofintravenously administering every 8 hours to a subject in need thereof apharmaceutical composition comprising 3.0 g of ceftolozane.

In another aspect, the invention provides a method of providingtazobactam or ceftolozane in the epithelial lining fluid of a subject inan amount effective to treat an intrapulmonary infection, comprising thestep of intravenously administering to the subject a pharmaceuticalcomposition comprising ceftolozane. The method may compriseadministering a pharmaceutical composition further comprisingtazobactam, optionally wherein the pharmaceutical composition isCXA-201. The method may comprise administering about 1.5 g ofceftolozane and tazobactam in total every 8 hours. In one embodiment,the amount of the ceftolozane in the ELF of the subject effective totreat an intrapulmonary infection is at least about 8 μg/ml. The ELFconcentration of ceftolozane in the ELF may reach at least about 8 μg/mlafter administration of the pharmaceutical composition. The subject istypically a human having, or believed to be at risk of having,nosocomial pneumonia. The subject (or patient) may, in some embodiments,have ventilator acquired pneumonia or hospital acquired pneumonia.

In another aspect, the invention provides the use of ceftolozane in themanufacture of a medicament for the treatment of an intrapulmonaryinfection comprising administering a therapeutically effective amount ofa pharmaceutical composition comprising the ceftolozane. The use maycomprise administering the pharmaceutical composition comprisingceftolozane, in combination with tazobactam.

In another aspect, the invention provides the use of ceftolozane in themanufacture of a medicament for the treatment of an intrapulmonaryinfection comprising intravenously administering a pharmaceuticalcomposition comprising 3.0 g of the ceftolozane every 8 hours to asubject in need thereof. The use may comprise administering thepharmaceutical composition comprising ceftolozane in combination withtazobactam. In one embodiment, the use comprises administeringceftolozane and tazobactam and the infection comprises Gram-negativebacteria.

In another aspect, the invention provides the use of ceftolozane in themanufacture of a medicament for the treatment of an intrapulmonaryinfection comprising intravenously administering a pharmaceuticalcomposition comprising the ceftolozane, wherein tazobactam orceftolozane is provided in the epithelial lining fluid of a subject inan amount effective to treat the intrapulmonary infection. The use maycomprise administering a pharmaceutical composition further comprisingtazobactam, optionally wherein the pharmaceutical composition isCXA-201. The use may comprise administering about 1.5 g of ceftolozaneand tazobactam every 8 hours. In one embodiment, the amount of theceftolozane in the ELF of the subject effective to treat anintrapulmonary infection is at least about 8 μg/ml. The ELFconcentration of ceftolozane in the ELF may reach at least about 8 μg/mlafter administration of the pharmaceutical composition. The subject istypically a human having, or believed to be at risk of having,nosocomial pneumonia. The subject (or patient) may, in some embodiments,have ventilator acquired pneumonia or hospital acquired pneumonia. Inthe methods and uses of the invention, the pharmaceutical compositionmay be administered parenterally. The pharmaceutical composition may beadministered intravenously. In some embodiments, the pharmaceuticalcomposition is intravenously administered about once every 8 hours as aninfusion. The pharmaceutical composition may be intravenouslyadministered as a 60-minute infusion.

In the methods and uses of the invention, the intrapulmonary infectionmay be an infection in the lung. The intrapulmonary infection may bepneumonia. In a preferred embodiment, the intrapulmonary infection isnosocomial pneumonia. The intrapulmonary infection may comprisePseudomonas aeruginosa, Enterobacteriaceae, or a combination thereof.Typically, the intrapulmonary infection comprises Pseudomonasaeruginosa. The intrapulmonary infection may comprise a pathogen withminimum inhibitory concentration for CXA-201 of ≦8 μg/ml. Theintrapulmonary infection may comprise a pathogen with minimum inhibitoryconcentration for ceftolozane of ≦8 μg/ml.

In another aspect, the invention provides ceftolozane, for use in amethod of treating an intrapulmonary infection. In one embodiment, theceftolozane is parenterally administered. Typically, the ceftolozane isintravenously administered. In some embodiments, the ceftolozane isadministered about once every 8 hours as an infusion. In someembodiments, the ceftolozane is intravenously administered as a60-minute infusion.

In one embodiment, the ceftolozane is for use in a method of treating anintrapulmonary infection wherein the intrapulmonary infection comprisesan infection in the lung. The intrapulmonary infection may be pneumonia.In a preferred embodiment, the ceftolozane is for use in a method oftreating nosocomial pneumonia. The intrapulmonary infection may comprisePseudomonas aeruginosa, Enterobacteriaceae, or a combination thereof.Typically, the intrapulmonary infection comprises Pseudomonasaeruginosa. The intrapulmonary infection may comprise a pathogen withminimum inhibitory concentration for ceftolozane and tazobactam of ≦8μg/ml. The intrapulmonary infection may comprise a pathogen with minimuminhibitory concentration for ceftolozane of ≦8 μg/ml.

The invention also provides ceftolozane, for use in a method of treatingan intrapulmonary infection, comprising administration of ceftolozane incombination with tazobactam. In one embodiment, the ceftolozane and/ortazobactam is parenterally administered. Typically, the ceftolozaneand/or tazobactam is intravenously administered. In some embodiments,the ceftolozane and/or tazobactam is administered about once every 8hours as an infusion. In some embodiments, the ceftolozane and/ortazobactam is intravenously administered as a 60-minute infusion. In oneembodiment, both the ceftolozane and tazobactam are parenterallyadministered. In another embodiment, both the ceftolozane and tazobactamare intravenously administered. In some embodiments, both theceftolozane and tazobactam are administered about once every 8 hours asan infusion. In some embodiments, both the ceftolozane and tazobactamare intravenously administered as a 60-minute infusion. In oneembodiment, the ceftolozane is for use in a method of treating anintrapulmonary infection wherein the intrapulmonary infection comprisesan infection in the lung. The intrapulmonary infection may be pneumonia.In a preferred embodiment, the ceftolozane is for use in a method oftreating nosocomial pneumonia. The intrapulmonary infection may comprisePseudomonas aeruginosa, Enterobacteriaceae, or a combination thereof.Typically, the intrapulmonary infection comprises Pseudomonasaeruginosa. The intrapulmonary infection may comprise a pathogen withminimum inhibitory concentration for ceftolozane and tazobactam of ≦8μg/ml. The intrapulmonary infection may comprise a pathogen with minimuminhibitory concentration for ceftolozane of ≦8 μg/ml.

In another aspect, the invention provides tazobactam, for use in amethod of treating an intrapulmonary infection, comprisingadministration of tazobactam in combination with ceftolozane. In oneembodiment, the tazobactam and/or ceftolozane is parenterallyadministered. Typically, the tazobactam and/or ceftolozane isintravenously administered. In some embodiments, the tazobactam and/orceftolozane is administered about once every 8 hours as an infusion. Insome embodiments, the tazobactam and/or ceftolozane is intravenouslyadministered as a 60-minute infusion. In one embodiment, both thetazobactam and ceftolozane are parenterally administered. In anotherembodiment, both the tazobactam and ceftolozane are intravenouslyadministered. In another embodiment, both the tazobactam and ceftolozaneare administered about once every 8 hours as an infusion. In anotherembodiments, both the tazobactam and ceftolozane are intravenouslyadministered as a 60-minute infusion.

In one embodiment, the tazobactam is for use in a method of treating anintrapulmonary infection wherein the intrapulmonary infection comprisesan infection in the lung. The intrapulmonary infection may be pneumonia.In a preferred embodiment, the tazobactam is for use in a method oftreating nosocomial pneumonia. The intrapulmonary infection may comprisePseudomonas aeruginosa, Enterobacteriaceae, or a combination thereof.Typically, the intrapulmonary infection comprises Pseudomonasaeruginosa. The intrapulmonary infection may comprise a pathogen withminimum inhibitory concentration for ceftolozane and tazobactam of ≦8μg/ml. The intrapulmonary infection may comprise a pathogen with minimuminhibitory concentration for ceftolozane of ≦8 μg/ml.

In another aspect, the invention provides ceftolozane and tazobactam, asa combined preparation for simultaneous, separate or sequential use in amethod of treating an intrapulmonary infection. In one embodiment, theceftolozane and tazobactam are parenterally administered. Typically, theceftolozane and tazobactam are intravenously administered. In someembodiments, the ceftolozane and tazobactam are administered about onceevery 8 hours as an infusion. In some embodiments, the ceftolozane andtazobactam, are intravenously administered as a 60-minute infusion.

In one embodiment, the ceftolozane and tazobactam are for use in amethod of treating an intrapulmonary infection wherein theintrapulmonary infection comprises an infection in the lung. Theintrapulmonary infection may be pneumonia. In a preferred embodiment,the ceftolozane and tazobactam are for use in a method of treatingnosocomial pneumonia. The intrapulmonary infection may comprisePseudomonas aeruginosa, Enterobacteriaceae, or a combination thereof.Typically, the intrapulmonary infection comprises Pseudomonasaeruginosa. The intrapulmonary infection may comprise a pathogen withminimum inhibitory concentration for ceftolozane and tazobactam of ≦8μg/ml. The intrapulmonary infection may comprise a pathogen with minimuminhibitory concentration for ceftolozane of ≦8 μg/ml.

In another aspect, the invention provides ceftolozane for use in amethod of providing tazobactam or ceftolozane in the epithelial liningfluid of a subject in an amount effective to treat an intrapulmonaryinfection, comprising the step of intravenously administeringceftolozane. In some embodiments, ceftolozane is administered incombination with tazobactam. Preferably, CXA-201 is administered. Inpreferred embodiments, about 1.5 g of ceftolozane and tazobactam isadministered every 8 hours. In one embodiment, the amount of theceftolozane in the ELF of the subject effective to treat anintrapulmonary infection is at least about 8 μg/ml. The ELFconcentration of ceftolozane in the ELF may reach at least about 8 μg/mlafter administration of the ceftolozane. The subject is typically ahuman having, or believed to be at risk of having, nosocomial pneumonia.The subject (or patient) may, in some embodiments, have ventilatoracquired pneumonia or hospital acquired pneumonia.

The safe and effective treatment of intrapulmonary infection withCXA-201 includes administration of an amount of the CXA-201 selected toprovide a therapeutically effective dose of the CXA-201 antibiotic inthe epithelial lining fluid (ELF). The penetration of CXA-201 into theELF compared to a piperacillin/tazobactam comparator was assessed in aPhase 1 clinical study in healthy adult volunteers. Thepiperacillin/tazobactam comparator contained piperacillin/tazobactam inan 8:1 weight ratio with a total of 2.79 mEq of sodium per gram ofpiperacillin, FDA approved under the tradename ZOSYN® (“Zosyn”). Thestudy results evaluate the penetration of intravenously administeredCXA-201 into healthy human lungs, as measured by the ratio of area underthe concentration-time curve (AUC) in epithelial lining fluid (ELF) toAUC in plasma (AUC(ELF)/AUC(plasma) ratio).

In the study, a 4.5 g amount of piperacillin/tazobactam incorporates thesame dose of tazobactam (0.5 g) as 1.5 g of CXA-201. A multiple-doseregimen was used in this study to ensure that the concentrations of theanalytes reached steady-state in both plasma and ELF prior toassessment. Healthy volunteers were chosen to standardize the subjectpopulation and minimize the variability associated with using activelyill patients. The objectives of the study included: (1) determinationand comparison of the ELF to plasma concentration ratios ofmultiple-doses of intravenous CXA-201 compared topiperacillin/tazobactam in healthy adult volunteers, and (2) assessmentof the safety and tolerability of multiple-doses of intravenous CXA-201in healthy adult volunteers.

The study was a Phase 1 prospective, randomized (1:1), comparatorcontrolled, open-label study of 50 healthy adult volunteers. Eachhealthy volunteer received 3 doses of either CXA-201 (1.5 grams every 8hours as a 60-minute infusion) or piperacillin/tazobactam (4.5 gramsevery 6 hours as a 30-minute infusion). Subjects received 3 doses of astudy drug, underwent serial blood draws at planned plasma samplingtimepoints, and underwent a single bronchoalveolar lavage (BAL)procedure at one of the scheduled timepoints (Table 1).

TABLE 1 Plasma Sampling and BAL Timepoints Plasma Sampling TimepointsBAL Timepoints Intensive plasma sampling from all 25 5 subjects pertimepoint subjects for one dosing interval per treatment group; in hoursfrom start of the third infusion CXA-201 0 (pre-PK dose trough), 1, 2,4, 6, 8 hours 1, 2, 4, 6, 8 hours post start post start of infusion ofthe third dose of of infusion of the third dose CXA 201 of CXA 201Piperacillin/tazobactam 0 (pre-PK dose trough), 0.5, 1, 2, 4, 6 hours0.5, 1, 2, 4, 6 hours post post start of infusion of the third dose ofstart of infusion piperacillin/tazobactam of the third dose ofpiperacillin/tazobactam

A total of 51 subjects were enrolled; 25 in the CXA-201 group and 26 inthe piperacillin/tazobactam group. Key Inclusion Criteria for the studywere: (1) healthy adult male or non-pregnant females between 18 and 50years, inclusive; (2) body mass index between 18.5 and 30; and (3)forced Expiratory Volume in 1 second (FEV1)≧80%. Key Exclusion Criteriafor the study were: (1) pregnancy or lactation; (2) clinicallysignificant systemic disease or the existence of any surgical or medicalcondition that may have interfered with the distribution, metabolism, orexcretion of CXA-201; (3) history of asthma or any restrictive orobstructive lung disease; (4) history of smoking or abuse of narcoticsor alcohol; (5) positive test for human immunodeficiency virus,Hepatitis B surface antigen, or Hepatitis C antibodies; (6) anycondition or situation where bronchoscopy was not advisable; and (7)impairment of renal function (CrCl<90 mL/min).

Determination of the ELF to Plasma Concentration Ratios ofMultiple-Doses of Intravenous CXA-201 Compared toPiperacillin/Tazobactam in Healthy Adult Volunteers.

Plasma and BAL datapoints were used to construct one concentration-timeprofile in the ELF using the mean concentrations at each time point.After dosing, a single ELF sample was obtained by bronchoalveolar lavage(BAL) from each healthy volunteer at one of 5 scheduled time points (5subjects/time point/treatment group). The ELF to plasma concentrationsof multiple-doses was determined Serial plasma samples were collectedpre- and post-treatment over a 6-hour (piperacillin/tazobactam) or8-hour (CXA-201) time period. Urea levels in the plasma and BAL wereused to calculate the ELF drug concentrations (see Table 1).Pharmacokinetic parameters for ELF were calculated by non-compartmentalanalysis using the mean concentrations at each time point. Theintrapulmonary penetration of CXA-201 into the ELF was determined bydividing the ELF AUC₀-t by mean plasma AUC₀-t.

The concentration of CXA-201 and piperacillin/tazobactam in ELF wereestimated from the concentration of drug in BAL fluid, the volume of BALfluid collected, and the ratio of urea concentration in BAL fluid tothat in plasma. Calculation of ELF volume was determined by the ureadilution method, using urea as an endogenous marker of ELF recovered byBAL. Concentration of CXA-201 and piperacillin/tazobactam in ELF wasestimated from the concentration of drug in BAL fluid, the volume of BALfluid collected, and the ratio of urea concentration in BAL fluid tothat in plasma. The following formulas represent these calculations:CXA-201(CXA/T)=[CXA/T]_(BAL) ×V _(BAL) /V _(ELF)

[CXA/T]_(BAL) is the concentration of CXA-201 in BAL fluid; V_(BAL) isthe volume of aspirated BAL fluid (total); Y_(ELF) isV_(BAL)×[urea]_(BAL)[urea]_(plasma), where [urea]_(BAL) is theconcentration of urea in the BAL fluid (supernatant) and [urea]_(plasma)is the concentration of urea in the plasma specimens.Piperacillin/tazobactam=[PIP/T]_(BAL) ×V _(BAL) /V _(ELF)

[PIP/T]BAL is the concentration of piperacillin/tazobactam in BAL fluid;V_(BAL) is the volume of aspirated BAL fluid (total); V_(ELF) isV_(BAL)×[urea]_(BAL)/[urea]_(plasma), where [urea]_(BAL) is theconcentration of urea in the BAL fluid (supernatant) and [urea]_(plasma)is the concentration of urea in the plasma specimens.

No oral antibiotic therapy was permitted. Safety was monitored throughthe review of vital signs, laboratory and physical examinations and theoccurrence of adverse events (AEs). Subjects who received three doses ofstudy medication and had both BAL and plasma samples collected wereincluded in the pharmacokinetic (PK) analysis population. All randomizedsubjects who received any dose (including partial doses) of studymedication were included in the safety analysis population.

The results of the study (Table 2) indicate that CXA-201 penetrated wellinto ELF. The ceftolozane component of CXA-201 ELF/plasma AUC ratio was0.48, compared to 0.26 for the piperacillin component ofpiperacillin/tazobactam. The ELF concentrations of ceftolozane exceeded8 μg/mL for 60% of the 8-hour dosing interval. The plasma concentrationsfor ceftolozane were consistent with those seen previously at this dose.

The ELF concentration vs. time profiles for ceftolozane and tazobactamcomponents of CXA-201 are shown in FIGS. 2A and 2B, respectively.Comparative data showing the ELF concentration vs. time profiles forpiperacillin and tazobactam components of the comparator drug are shownin FIGS. 3A and 3B, respectively. The ELF to plasma penetration ratiosare shown in Table 2.

The PK parameters were determined by non-compartmental PK analysis.PHOENIX® WinNonlin v 6.1 (PHARSIGHT®, Mountain View, Calif.) was usedfor the derivation of all PK individual measures for each subject. ThePK parameters for ELF were calculated by taking the mean concentrationsof the 5 subjects at each time point and constructing a single profileover the duration of sampling. In the event that the urea concentrationsdetermined in plasma or ELF were below quantifiable limits, therebyproviding only an estimate of concentration, those values were not usedin the calculation of mean concentration at that time point. Theceftolozane, piperacillin, and tazobactam PK parameters that werecomputed in plasma and ELF were:

-   -   C_(max) (μg/mL): Maximum plasma and ELF concentration over the        entire sampling phase directly obtained from the experimental        plasma concentration time data, without interpolation.    -   T_(max) (hr): Sampling time at which C_(max) occurred, obtained        directly from the experimental plasma and ELF concentration time        data, without interpolation.    -   C_(last) (μg/mL): Plasma or ELF concentration when last        quantifiable concentration was observed, relative to the end of        infusion.    -   T_(last) (hr): Time when the last quantifiable concentration was        observed.    -   AUC_(0-t) (μg*hr/mL): An area under the concentration time curve        from the time of the dose to the end of the dosing interval.    -   Percent penetration into ELF: Calculated as the ratio of        AUC_(0-tELF) and mean AUC_(0-tPlasma).

TABLE 2 Summary of ELF to Plasma Penetration Ratios Mean Plasma ELFAUC_(0-τ) ELF AUC_(0-τ) Penetration Analyte (μg * hr/mL) (μg * hr/mL)Ratio ceftolozane 158.5 75.1 0.48 (in CXA-201) Tazobactam 19.3 8.5 0.44(in CXA-201) Piperacillin 357.3 94.5 0.26 (in piperacillin/tazobactam)Tazobactam 46.1 24.7 0.54 (in piperacillin/tazobactam)

The ELF/plasma AUC ratio for the ceftolozane component of CXA-201 was0.48, compared to 0.26 for the piperacillin component of the comparatordrug (piperacillin/tazobactam). The ELF/plasma AUC ratio for tazobactamwas 0.44 and 0.54 when given as part of CXA-201 andpiperacillin/tazobactam, respectively. The ELF concentrations ofceftolozane exceeded 8 μg/mL for 60% of the 8-hour dosing interval. Theplasma and ELF concentrations of tazobactam when given aspiperacillin/tazobactam was approximately 2-fold higher than when anequivalent dose was given as CXA-201.

The results show that ceftolozane and tazobactam (i.e., administered asCXA-201) penetrated well into the ELF of healthy volunteers compared topiperacillin/tazobactam, an agent widely used for treatment of lowerrespiratory infections. CXA-201's intrapulmonary pharmacokineticssupport use of CXA-201 as a parenteral (e.g., intravenous) antibioticfor treatment of lower respiratory tract infections, includinginfections caused by pathogens with minimum inhibitory concentrations of≦8 μg/ml. The concentrations of ceftolozane in ELF exceeded 8 μg/mL, aconcentration that inhibits 99% of P. aeruginosa, for approximately 60%of the 8-hour dosing interval for the CXA-201 regimen of 1.5 grams everyeight hours as a 60 minute infusion.

Assessment of the Safety and Tolerability of Multiple-Doses ofIntravenous CXA-201 in Healthy Adult Volunteers

Among the subjects, 50 of the 51 (98%) subjects received all 3 doses ofstudy medication and completed the BAL procedure. One subjectprematurely discontinued piperacillin/tazobactam and terminated theirparticipation in the study due to an AE of hypersensitivity thatoccurred during administration of the first dose. Demographics andbaseline characteristics are summarized in Table 3, the two treatmentarms were well balanced.

TABLE 3 Demographics and Baseline Characteristics (Safety Population)Piperacillin/ CXA-201 tazobactam 1.5 grams 4.5 grams (N = 25) (N = 26)Sex, n (%) Female 11 (44.0) 11 (42.3) Male 14 (56.0) 15 (57.7) Age,years Mean (SD) 32.6 (7.8)   34.2 (8.5)   Minimum, Maximum 21, 47 22, 49Race, n (%) White 20 (80.0) 21 (80.8) Black or African American 2 (8.0)2 (7.7) Asian 1 (4.0) 0 (0.0) American Indian or Alaska Native 0 (0.0) 1(3.8) Native Hawaiian or Pacific Islander 1 (4.0) 0 (0.0) Other 1 (4.0)2 (7.7) BMI, kg/m² Mean (SD) 26.21 (2.6)    23.23 (2.4)    Minimum,Maximum 22.3, 30.0 20.6, 29.9

During the study, treatment-emergent AEs (TEAEs) occurred in 20.0%(5/25) of subjects receiving CXA-201 and 23.1% (6/26) of subjectsreceiving piperacillin/tazobactam. No serious AEs were reported ineither treatment group. All AEs were mild in severity. The incidence andpattern of AEs were generally similar in the 2 treatment groups, Table4.

TABLE 4 TEAEs by Preferred Term (Safety Population) Subjects with atleast 1 TEAE  5 (20.0)  6 (23.1) Diarrhea 1 (4.0)  3 (11.5) Viral UpperRespiratory Infection 1 (4.0) 0 (0)   Musculoskeletal Chest Pain 1 (4.0)0 (0)   Somnolence 1 (4.0) 0 (0)   Hematuria 1 (4.0) 0 (0)   Cough 1(4.0) 0 (0)   Type I Hypersensitivity 0 (0)   1 (3.8) AlanineAminotransferase Increased 0 (0)   1 (3.8) Aspartate AminotransferaseIncreased 0 (0)   1 (3.8) Blood Creatine Phosphokinase Increased 0 (0)  1 (3.8) Hyperkalemia 0 (0)   1 (3.8)

Eight subjects had TEAEs assessed as related to study drug; two in theCXA-201 group (diarrhea and somnolence in 1 subject each) and six in thepiperacillin/tazobactam group (diarrhea in 3 subjects, type Ihypersensitivity in 1 subject, blood creatine phosphokinase increased in1 subject, and alanine aminotransferase increased, aspartateaminotransferase increased, and hyperkalaemia all in the same 1subject). One piperacillin/tazobactam-treated subject discontinued studydrug due to an adverse event, type I hypersensitivity. There were noclinically significant changes in safety laboratory assessments or vitalsigns.

CXA-201 appeared safe and well tolerated in this group of healthy adultsubjects.

Determining Appropriate Dose

A Monte Carlo simulation was performed based on clinical trial data topredict an effective CXA-201 dose for treating nosocomial pneumoniausing PHOENIX® NLME (PHARSIGHT®, Mountain View, Calif.) software, a toolfor data processing and modeling for population PK/PD analysis. Apopulation pharmacokinetic (PK) model was developed using the CXA-201plasma concentration versus time data from a previously conducted Phase2 study in patients with complicated intra abdominal infections.Estimates of clearance and volume of distribution along with theassociated inter-individual variability were obtained from theseanalyses. The outputs from the PK population model served as inputs fora clinical trial simulation performed using PHARSIGHT® Trial Simulator(PHARSIGHT®) software, a tool for defining and testing interactive drugmodels, exploring and communicating study design attributes, andperforming statistical and sensitivity analysis through graphical andstatistical summaries. Based on the mean ELF penetration data, anELF/Plasma AUC ratio of 0.48 for ceftolozane (modeled as a numericalrange of 0.25-0.65) calculated from the ceftolozane ELF study mentionedabove was used to generate a random/Plasma AUC ratio from the range0.25-0.65 for each simulated patient. This range reflects a conservativeestimate of the potential distribution in a patient population. Usingthe results from the PK population model and the ELF/Plasma AUC ratio,the model simulated plasma and ELF concentration of CXA-201 versus timeprofiles for 1,000 hypothetical clinical trial patients with nosocomialpneumonia. The model evaluated the probability of clinical success ofthe 3.0 g every 8 hour (q8 h) dose of CXA-201 against three keypathogens in nosocomial pneumonia. The MIC distribution for thesepathogens was imputed from 2008 United States surveillance data.Clinical success was defined as the achievement of an ELF or plasmaconcentration of ceftolozane higher than the MIC(s) of the lowerrespiratory pathogen(s) for a given patient. In vivo models havedemonstrated that, as for typical cephlaosporins, the relavent PK/PDdriver for CXA-201 is the percentage of time above MIC during the dosinginterval. The target is to achieve concentrations that exceed the MIC ofthe pathogen for 45-50% of the time between each q8H dose. Thus, athreshold of 50% time above the minimum inhibitory concentration [T>MIC]on Day 7 of treatment was used. Plasma and ELF concentrations wereestimated at 15 time-points post-administration on Day 7 when dosedevery 8 hours. The results of these simulations are shown in Table 5.

TABLE 5 Probability of Target Attainment versus Key Pathogens inNosocomial Pneumonia Using the Simulated 3.0 g versus the 1.5 g Dose ofCeftolozane/tazobactam 50% T > MIC in 50% T > MIC Pathogen DosingRegimen Plasma in ELF P. aeruginosa 1.5 g q8h 98.2 94.6 3.0 g q8h 99.498.5 E. coli 1.5 g q8h 96.3 94.2 3.0 g q8h 98.8 95.5 K. pneumoniae 1.5 gq8h 90.2 87.3 3.0 g q8h 92.6 89.3 Abbreviation: T > MIC = Time aboveminimum inhibitory concentration.These simulations demonstrate that the 3.0 g dose of CXA-201administered every 8 hours is expected to provide adequateconcentrations for treatment of the vast majority of lower respiratoryinfections caused by these pathogens.

Following these simulations, the safety and tolerability of a 10 daycourse of CXA-201 3.0 g IV q8 h was evaluated in healthy humanvolunteers. Subjects were randomized to receive either 3.0 g (2.0/1.0 g)CXA-201 (n=8), 1.5 g (1.0/0.5 g) CXA-201 (n=4), or placebo (n=4). Thedata showed that CXA-201 was generally safe and well tolerated in thisstudy. There were no serious adverse events or deaths reported in thisstudy.

In conclusion, given the pharmacokinetic simulations conducted, thefavorable data from the intrapulmonary PK study and demonstrated safetyand tolerability of the higher dose of CXA-201 in the Phase 1 studymentioned above, the data provide justification for the use of 3.0 gCXA-201 IV q8 h for the treatment of patients with nosocomial pneumoniacaused by Gram-negative pathogens.

Preparing CXA-201

CXA-201 can be prepared by combining ceftolozane and tazobactam in a 2:1weight ratio. CXA-201 can be obtained using methods described in U.S.Pat. No. 7,129,232 and Toda et al., “Synthesis and SAR of novelparenteral anti-pseudomonal cephalosporins: Discovery of FR264205,”Bioorganic & Medicinal Chemistry Letters, 18, 4849-4852 (2008),incorporated herein by reference in its entirety.

According to the method disclosed in Toda et al., “Synthesis and SAR ofnovel parenteral anti-pseudomonal cephalosporins: Discovery ofFR264205,” Bioorganic & Medicinal Chemistry Letters, 18, 4849-4852(2008), ceftolozane can be obtained by the synthetic schemes of FIGS. 4Aand 4B. Referring to FIGS. 4A and 4B, synthesis of ceftolozane can beperformed via activation of the thiadiazolyl-oximinoacetic acidderivative (I) with methanesulfonyl chloride and K₂CO₃ in DMA at 10° C.,followed by coupling with the 7-aminocephem (II) by means of Et₃N incold EtOAc/H₂O, affords amide (III) (1). Substitution of the allylicchloride of compound (III) with4-RN-Boc-aminoethyl)carbamoylaminol-1-methyl-5-tritylaminopyrazole (IV)in the presence of 1,3-bis(trimethylsilyl)urea (BSU) and KI in DMF thenaffords the protected pyrazolium adduct (V), which, after fulldeprotection with trifluoroacetic acid in anisole/CH₂Cl₂, can beisolated as the hydrogensulfate salt by treatment with H2SO4 ini-PrOH/H₂O (1, 2). Scheme 1. The pyrazolyl urea intermediate (IV) can beprepared as follows. Treatment of 5-amino-1-methylpyrazole (VI) withNaNO₂/HCl in water at 5° C. gives the 4-nitrosopyrazole derivative(VII), which can be reduced to the diaminopyrazole (VIII) by catalytichydrogenation over Pd/C in the presence of H₂SO₄. Selective acylation ofthe 4-amino group of compound (VIII) with phenyl chloroformate in thepresence of NaOH in H₂O/dioxane at 10° C. then yields the phenylcarbamate (IX). After protection of the free amine group of carbamate(IX) with chlorotriphenylmethane in the presence of Et₃N in THF, theresulting N-trityl derivative (X) can be coupled withN-Boc-ethylenediamine (XI) in the presence of Et₃N in DMF to affordpyrazolyl urea (IV).

Biological Activity Assay

The antibacterial activity of the CXA-201 or other compounds can bemeasured by the minimum inhibitory concentrations (MIC) of the compoundsagainst various bacteria measured by using the broth microdilutionmethod performed according to Clinical and Laboratory StandardsInstitute (CLSI) guidelines with modifications as described below (CLSIguidelines can be derived from the CLSI document M7-A8 published inJanuary 2009: “Methods for Dilution Antimicrobial Susceptibility Testsfor Bacteria That Grow Aerobically; Approved Standard-Eighth Edition”).

To prepare for MIC testing, individual colonies can be isolated bystreaking frozen glycerol material containing Staphylococcus orPseudomonas spp. onto rich, non-selective, tryptic soy agar containing5% sheep's blood (TSAB), and incubated at 37° C. for 18-24 hrs.

On the day of testing, primary cultures can be started by scraping off5-10 colonies from the TSAB plates. The material can be suspended in ˜5mL of cation adjusted Mueller Hinton Broth (CAMHB) in 14 mL culturetubes and can be incubated at 37° C. with aeration (200 rpm) for ˜2 hrsuntil the OD600 was ≧0.1.

Inoculum cultures can be prepared by standardizing the primary culturesto OD600=0.1 and then adding 20 μL of the adjusted primary culture per 1mL CAMHB for Pseudomonas and CAMHB plus 4% NaCl for MRSA so that thefinal inoculum density was ˜10⁵ colony forming units per milliliter.Diluted inoculum cultures can be used to inoculate 50 μL per well in 96well broth microdilution assay plates. 50 μL of CAMHB that containedcompound concentrations ranging from 64-0.06 μg/mL in two-fold dilutionscan also be added to the broth microdilution assay plates for a finalvolume 100 μL per well, therefore final culture OD₆₀₀ was approximately0.001 and the final NaCl concentration for the MRSA strain was 2%.

Plates can be incubated for 18-20 hours at 37° C. with aeration (200rpm). Following incubation, growth can be confirmed visually placingplates over a viewing apparatus (stand with a mirror underneath) andthen OD₆₀₀ can be measured using a SpectraMax 340PC384 plate reader(Molecular Devices, Sunnyvale, Calif.). Growth was defined as turbiditythat could be detected with the naked eye or achieving minimum OD₆₀₀ of0.1. MIC values were defined as the lowest concentration producing novisible turbidity.

The examples and illustrative embodiments described herein are providedby way of illustration, and do not constitute additional limitations onthe scope of the claims. While some embodiments have been shown anddescribed in the instant specification, the specification as ready byone of ordinary skill in the relevant arts also discloses variousmodifications and substitutions of embodiments explicitly disclosedherein. The exemplary embodiments from the specification are notprovided to read additional limitations into the claims.

The invention claimed is:
 1. A method of treating an infection selectedfrom the group consisting of: nosocomial pneumonia, ventilator acquiredpneumonia and hospital acquired pneumonia, the method comprisingrepeatedly intravenously administering 2.0 g of ceftolozane and 1.0 g oftazobactam to a subject in need thereof about once every 8 hours.
 2. Themethod of claim 1, wherein the method comprises administering theceftolozane as a 60-minute infusion.
 3. The method of claim 1, whereinthe ceftolozane is a hydrogen sulfate salt.
 4. The method of claim 1,wherein the ceftolozane and tazobactam are administered as a singlepharmaceutical composition.
 5. The method of claim 4, wherein the methodcomprises administering the pharmaceutical composition as a 60-minuteinfusion.
 6. The method of claim 1, wherein the pneumonia is nosocomialpneumonia.
 7. The method of claim 1, wherein the pneumonia is ventilatoracquired pneumonia.
 8. The method of claim 1, wherein the pneumonia ishospital acquired pneumonia.
 9. The method of claim 1, wherein theceftolozane is administered in its free base form.
 10. The method ofclaim 1, wherein the ceftolozane is administered in its salt form. 11.The method of claim 6, wherein the ceftolozane is administered in itsfree base form.
 12. The method of claim 6, wherein the ceftolozane isadministered in its salt form.
 13. The method of claim 1, wherein theinfection comprises one or more pathogens selected from the groupconsisting of: Pseudomonas aeruginosa, E. coli and K. pneumoniae.